A number of lipid-based materials such as liposomes have been used as biological carriers for many pharmaceutical and other biological applications, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcriptional factors and other cellular vectors into a variety of cultured cell lines and animals. Clinical trials have demonstrated the effectiveness of liposome-mediated drug delivery for targeting liposome-entrapped drugs to specific tissues and specific cell types. See, for example, U.S. Pat. No. 5,264,618, which describes techniques for using lipid carriers, including the preparation of liposomes and pharmaceutical compositions and the use of such compositions in clinical situations.
More recently, cationic lipids have been used to deliver nucleic acids to cells, allowing uptake and expression of foreign genes. In particular, cationic lipid-mediated delivery of exogenous nucleic acids in vivo in humans and/or various commercially important animals will ultimately permit the prevention, amelioration and cure of many important diseases and the development of animals with commercially important characteristics. The exogenous genetic material, either DNA or RNA, may provide a functional gene which, when expressed, produces a protein lacking in the cell or produced in insufficient amounts, or may provide an antisense RNA or ribozyme to interfere with a cellular function in, e.g., a virus-infected cell or a cancer cell, thereby providing an effective therapeutic for a disease state.
Nucleic acids are generally large polyanionic molecules which, therefore, bind cationic lipids through charge interactions. While lipid carriers have been shown to enhance nucleic acid delivery in vitro and in vivo, the mechanism by which they facilitate transfection is not clearly understood. While it was initially believed that lipid carriers indicated transfection by promoting fusion with plasma membranes, allowing delivery of the DNA complex into the cytoplasm, it is now generally accepted that the primary mechanism of cellular uptake is by endocytosis.
While the mechanism by which cationic lipid carriers act to mediate transfection is not clearly understood, they are postulated to act in a number of ways with respect to both cellular uptake and intracellular trafficking. Some of the proposed mechanisms by which cationic lipids enhance transfection include: (i) compacting the DNA, protecting it from nuclease degradation and enhancing receptor-mediated uptake, (ii) improving association with negatively-charged cellular membranes by giving the complexes a positive charge, (iii) promoting fusion with endosomal membranes facilitating the release of complexes from endosomal compartments, and (iv) enhancing transport from the cytoplasm to the nucleus where DNA may be transcribed. When used for in vivo delivery, the role of the cationic lipid carriers is further complicated by the interactions between the lipid-nucleic acid complexes and host factors, e.g., the effects of the lipids on binding of blood proteins, clearance and/or destabilization of the complexes.
Typically, cationic lipids are mixed with a non-cationic lipid, usually a neutral lipid, and allowed to form stable liposomes, which liposomes are then mixed with the nucleic acid to be delivered. The liposomes may be large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). The liposomes are mixed with nucleic acid in solution, at concentrations and ratios optimized for the target cells to be transfected, to form cationic lipid-nucleic acid transfection complexes. Alterations in the lipid formulation and mode of delivery allow preferential delivery of nucleic acids to particular tissues in vivo. PCT patent application numbers WO 96/40962, WO 96/40963.
The majority of studies on cationic lipid-mediated delivery have focused on the cationic lipid component, with relatively little work aimed at the role of non-cationic co-lipids (also called helper lipids). A commonly used helper lipid is dioleoylphosphatidylethanolamine (DOPE). DOPE was shown to improve transfection efficiencies in vitro when used in conjunction with a number of different cationic lipids. Felgner et al., (1994) Proc. Natl. Acad. Sci. (USA) 269(4):2550-2561. It has been commonly believed that DOPE improved transfection by making the liposomes more fusogenic, thereby improving either fusion with the plasma membrane, fusion with the endosomal membrane, or both. However, the studies describing the role of DOPE as a neutral lipid were performed in vitro and did not address its effect in vivo. Other studies have shown that in vitro transfection results are not predictive of in vivo transfection and, therefore, lipid formulations that were optimized for in vitro transfection were not necessarily optimal in vivo. Recent reports have obtained improved in vivo transfection efficiencies using cholesterol as the helper lipid. Liu et al., (1997) Nature Biotech. 15: 167-173; Solodin et al., (1995) Biochem. 34(41): 13537-13544.
While the use of cationic lipid carriers for transfection is well-known, structure activity relationships are not well understood. It is postulated that different lipid carriers will affect each of the various steps in the transfection process (e.g., condensation, uptake, nuclease protection, endosomal release, nuclear trafficking, and decomplexation) with greater or lesser efficiency, thereby making the overall transfection rate difficult to correlate with lipid structures. Thus, alterations in either the cationic or helper lipid component do not have easily predictable effects on activity. For the most part, therefore, improvements to known cationic lipid-mediated delivery systems are dependent on empirical testing. When intended for in vivo transfection, new lipids and lipid formulations should be screened in vivo to accurately predict optimal lipids and formulations for transfection of target cells.
It is desirable to have improved lipid delivery systems, e.g., to achieve higher levels of in vivo gene transfection. Improved levels of gene transfection will allow the treatment of disease states for which higher levels of expression are needed for therapeutic effect than achievable with prior art lipid delivery systems. Alternatively, higher transfection levels allow use of smaller amounts of material to achieve comparable expression levels, thereby decreasing potential lipid-associated toxicities and decreasing cost. Further, by choice of neutral lipid, the toxicity of particular cationic lipids can be decreased. The present invention provides these and related advantages as well.